Systematics and signalling of Madagascan chameleons of the Calumma nasutum group

Background Madagascar is famous for its biodiversity and exceptional degree of endemism, especially in chameleons, hosting almost half of the world’s species. The exploration of its species diversity started with the famous Georges Cuvier who described the first Madagascan chameleons in 1829. Since then, the quality of species descriptions and, subsequently, the species concepts have substantially changed. While the first descriptions were based on a few characters of the external morphology only, today the collected specimens are usually DNA barcoded and their taxonomic status is evaluated based on genetic distances as a first step. In doing so, a previous molecular study of the Calumma nasutum group, which are small chameleons with a rostral appendage on the snout tip in most species, resulted in an impressive 33 deep mitochondrial lineages. Only seven of these corresponded to named species leaving 26 lineages as operational taxonomic units (OTUs). In times of DNA based taxonomy and species delimitation algorithms taxonomists face new challenges of how to describe a species and to avoid oversplitting. In this dissertation I evaluate, based on a taxonomic revision of Calumma nasutum group, the significance of mitochondrial lineages for species delimitation and promote micro-computed tomography (micro-CT) as an additional tool for integrative taxonomy. The second part deals with the discovery of the phenomenon of widespread fluorescence in chameleons. Biofluorescence is only rarely found in land vertebrates, so far, but has been reported for several marine organisms where it is used, inter alia, for intraspecific communication. As chameleons also communicate visually, the fluorescent pattern might work as an additional signal for species recognition. Methods and Procedure To describe (or redescribe) species of the Calumma nasutum group, I followed an integrative taxonomic approach incorporating five lines of evidence (for details, see below): External Morphology, micro-CT-scans of the skulls, dice-CT scans of the hemipenes, mitochondrial gene sequences (ND2), and nuclear gene sequences (CMOS). In the Calumma nasutum group a number of species have remained poorly characterized, because their original descriptions date to over a century ago and lack precise locality data, or because the holotype is a juvenile specimen. These species were redescribed here using a combination of micro-CT scans and detailed study of external morphology. With the help of diagnostic characters of the skull, the old type specimens were matched to recently collected and sequenced specimens. Additionally, micro-CT scanning was used for the first time to produce 3-dimensional models of chameleon hemipenes. In preparation for scanning, each hemipenis was removed from the specimen and immersed in iodine solution for several days to enhance the contrast when X-raying this soft tissue. This method is called dice-CT, and the resulting scans provide a more objective and detailed illustration of the hemipenes than the conventional 2-dimensional drawings. Further, sequences of mitochondrial (ND2) and nuclear (CMOS) genes were analysed for most of the new species described here. To provide comparability, access, and fast taxonomic progress, all new species were registered at ZooBank with an LSID number, their sequences were uploaded to GenBank, and all taxonomic acts were published in open access journals. To study fluorescence in chameleons, we used a fluorimeter to measure the excitation and emission spectra and to calculate the quantum yield for the intensity of fluorescence. The distribution of the fluorescent tubercles was recorded with photographs under UV light illumination, and the bony origins of fluorescence were studied using micro-CT and Transmission Electron microscopy of histological sections. Conclusions The taxonomic part or this dissertation resulted in the description of eight new chameleon species, "Calumma emelinae", C. gehringi, C. juliae, C. lefona, "C. ratnasariae", C. roaloko, "C. tjiasmantoi", and C. uetzi (see chapter 3.1.2, 3.1.3, 3.1.4, 3.1.5), contributing nearly 4% of all known species of the family Chamaeleonidae. Furthermore, one species, C. radamanus (Mertens, 1933) was revalidated, five species, C. boettgeri (Boulenger, 1888), C. fallax (Mocquard, 1900), C. guibei (Hillenius, 1959), C. linotum (Müller, 1924), and C. nasutum (Duméril & Bibron, 1836) were redescribed, and the females of C. vatosoa Andreone, Mattioli, Jesu & Randrianirina, 2001 were described for the first time. Using an integrative taxonomic approach, I showed that current species delimitation algorithms based on mitochondrial gene sequences alone greatly overestimate the actual number of species. Micro-CT proved essential for analysing skull morphology, which resulted more appropriate for species delimitation than some highly variable external characters. Further, this tool enabled to find frontoparietal fenestrae, which are cranial openings of potential adaptive importance found only in chameleon species living at high elevations. Using novel dice-CT imaging, I also analysed hemipenial morphology in minute detail and described a new ornament, the “cornucula gemina”. Finally, the phenomenon of fluorescence in chameleons was discovered in species belonging to eight of the twelve chameleon genera. The optimal excitation wavelength is in the UV-A spectrum at 353 nm, emitting light with wavelengths from 360 nm to 500 nm, with a maximum at 433 nm (blue spectrum). We showed that the fluorescent patterns result from bony tubercles on the skull are species specific, sexually dimorphic, and occur especially in forest living species. Based on these findings, and also because the colour blue is a conspicuous signal in forest habitats, I hypothesize that chameleons use fluorescence as a constant signal for intraspecific communication supplementing their vibrant body-colour language. Further, as-of-yet unpublished examples suggest that fluorescence is more common in squamates and might be an interesting field for further studies.Hintergrund Madagaskar ist bekannt für seine Biodiversität und seine außergewöhnlich hohe Endemismusrate. Dies gilt vor allem für Chamäleons, da auf der Insel beinahe die Hälfte aller Arten weltweit vorkommt. Die Erforschung dieser Artenvielfalt begann mit dem berühmten Georges Cuvier, der die ersten madagassischen Chamäleons im Jahre 1829 beschrieb. Seit damals haben sich die Qualität der Artbeschreibungen und folglich auch das Artkonzept substanziell verändert. Beruhten die ersten Beschreibungen noch auf wenigen Merkmalen der äußeren Morphologie, so werden heutzutage die gesammelten Exemplare gewöhnlich erst sequenziert und ihre Artzugehörigkeit anhand von genetischen Abständen bewertet. Auf diese Weise stellte eine vorangegangene Studie fest, dass die Calumma-nasutum-Gruppe, die aus kleinen Chamäleons mit gewöhnlich einem Nasenfortsatz auf der Schnauze besteht, insgesamt 33 tiefe mitochondriale Linien enthält. Nur sieben davon gehörten zu bereits beschriebenen Arten, wodurch noch 26 Linien als operational taxonomic units (OTUs) übrig blieben. In Zeiten der DNA-geleiteten Taxonomie und der Verwendung von Algorithmen zur Artabgrenzung stehen Taxonomen neuen Herausforderungen gegenüber, eine Art zu beschreiben und ein sogenanntes „oversplitting“ zu vermeiden. In dieser Dissertation bewerte ich am Beispiel der Revision der Calumma-nasutum-Gruppe die Bedeutung von mitochondrialen Linien zur Artabgrenzung und stelle Mikrocomputertomografie (Mikro-CT) als zusätzliches Werkzeug der integrativen Taxonomie vor. Der zweite Teil handelt von der Entdeckung des Phänomens der weitverbreiteten Fluoreszenz bei Chamäleons. Biofluoreszenz wurde bisher nur selten bei Landvertebraten nachgewiesen, ist jedoch bei einigen Meeresorganismen gut bekannt, welche die Fluoreszenz unter anderem zur intraspezifischen Kommunikation nutzen. Da Chamäleons ebenfalls optisch kommunizieren, könnten die Fluoreszenzmuster als zusätzliches Signal zur Arterkennung dienen. Methoden und Vorgehensweise Um Arten der Calumma-nasutum-Gruppe (wieder) zu beschreiben, nutzte ich einen integrativen taxonomischen Ansatz mit fünf Beweislinien (siehe unten im Detail): Äußere Morphologie, Mikro-CT-Scans der Schädel, dice-CT-Scans der Hemipenisse, Sequenzen der mitochondrialen Gene und Sequenzen der Kerngene. Einige Arten der Calumma-nasutum-Gruppe waren bisher wenig charakterisiert, da ihre Originalbeschreibung über einhundert Jahre zurücklag und genaue Typuslokalitäten fehlten oder weil der Holotypus ein Jungtier ist. Diese Arten wurden unter Verwendung von mikro-CT-Scans und genauer Untersuchung der äußeren Morphologie wiederbeschrieben. Mit Hilfe diagnostischer Merkmale des Schädels wurden die alten Holotypen kürzlich gesammelten und sequenzierten Individuen zugeordnet. Zusätzlich wurden zum ersten Mal Mikro-CT-Scans genutzt, um dreidimensionale Modelle von Chamäleonhemipenissen zu entwerfen. Zur Vorbereitung des Scans wurde jeder Hemipenis vom Tier abgetrennt und in Lugolscher Lösung für mehrere Tage eingelegt, um den Kontrast dieses weichen Gewebes beim Röntgen zu erhöhen. Diese Methode nennt sich dice-CT und die resultierenden Scans bieten eine objektivere und detailliertere Darstellung des Hemipenis als die herkömmlichen zweidimensionalen Zeichnungen. Weiterhin wurden Sequenzen von mitochondrialen Genen (ND2) und Kerngenen (CMOS) für die meisten der hier neu beschriebenen Arten analysiert. Um Vergleichbarkeit, weltweiten Zugang und schnellen taxonomischen Fortschritt zu garantieren, wurden alle neuen Arten bei ZooBank unter einer LSID-Nummer registriert, ihre Sequenzen bei GenBank hochgeladen und alle taxonomischen Arbeiten in open-access-Journalen veröffentlicht. Zur Untersuchung der Fluoreszenz bei Chamäleons benutzten wir ein Fluorimeter, um die Anregungs- und Emissionsspektren zu messen und die Quantenausbeute zu berechnen. Die Verteilung der fluoreszierenden Tuberkel wurde mit Fotos unter UV-Beleuchtung dokumentiert und ihr knöcherner Ursprung mit Mikro-CT und Transmissionselektronenmikroskopie der histologischen Schnitten untersucht. Schlussfolgerungen Im taxonomischen Teil dieser Dissertation wurden acht Chamäleonarten neu beschrieben, die beinahe 4% aller bisher bekannten Arten der Familie Chamaeleonidae ausmachen: "Calumma emelinae", C. gehringi, C. juliae, C. lefona, "C. ratnasariae", C. roaloko, "C. tjiasmantoi", und C. uetzi (siehe Kapitel 3.1.2, 3.1.3, 3.1.4, 3.1.5). Weiterhin wurde eine Art, C. radamanus (Mertens, 1933), revalidiert. Es wurden fünf Arten wiederbeschrieben: C. boettgeri (Boulenger, 1888), C. fallax (Mocquard, 1900), C. guibei (Hillenius, 1959), C. linotum (Müller, 1924), und C. nasutum (Duméril & Bibron, 1836), und die Weibchen von C. vatosoa Andreone, Mattioli, Jesu & Randrianirina, 2001 wurden erstmals beschrieben. Mit Verwendung eines integrativ taxonomischen Ansatzes konnte ich zeigen, dass Algorithmen zur Artabgrenzung, die allein auf mitochondrialen Gensequenzen basieren, die eigentliche Anzahl an Arten deutlich überschätzen. Mikro-CT bewährte sich um die Schädelmorphologie zu untersuchen, welche sich besser zur Artabgrenzung eignete als die sehr variablen äußeren Merkmale. Außerdem konnten mit Hilfe dieses Werkzeugs auch Frontoparietalfenster gefunden werden. Diese Schädelöffnungen wurden nur bei Chamäleons aus Montangebieten nachgewiesen und könnten eine besondere Anpassung an den Lebensraum darstellen. Das neuartige Dice-CT-Verfahren ermöglichte die Hemipenismorphologie im kleinsten Detail zu untersuchen und auch ein neues Ornament, die “cornucula gemina”, zu beschreiben. Schließlich wurde bei Arten aus acht der zwölf Chamäleongattungen das Phänomen der Fluoreszenz entdeckt. Die optimale Anregungswellenlänge liegt im UV-A-Spektrum bei 353 nm und Licht mit Wellenlängen von 360 bis 500 nm, mit einem Maximum bei 433 nm im blauen Spektrum, wird emittiert. Wir konnten zeigen, dass die Fluoreszenzmuster von knöchernen Tuberkeln des Schädels stammen, artspezifisch und sexualdimorph sind und vor allem bei waldbewohnenden Arten vorkommen. Darauf aufbauend und auch, da die Farbe Blau im Wald ein auffallendes Signal darstellt, formuliere ich die Hypothese, dass Chamäleons die Fluoreszenz als konstantes Signal zur intraspezifischen Kommunikation nutzen und damit ihre Farbensprache ergänzen. Nicht-publizierte Beispiele lassen außerdem vermuten, dass Fluoreszenz bei Squamaten weiter verbreitet ist und ein interessantes Gebiet für nachfolgende Studien darstellen könnte

Systematics and signalling of Madagascan chameleons of the Calumma nasutum group

Background Madagascar is famous for its biodiversity and exceptional degree of endemism, especially in chameleons, hosting almost half of the world’s species. The exploration of its species diversity started with the famous Georges Cuvier who described the first Madagascan chameleons in 1829. Since then, the quality of species descriptions and, subsequently, the species concepts have substantially changed. While the first descriptions were based on a few characters of the external morphology only, today the collected specimens are usually DNA barcoded and their taxonomic status is evaluated based on genetic distances as a first step. In doing so, a previous molecular study of the Calumma nasutum group, which are small chameleons with a rostral appendage on the snout tip in most species, resulted in an impressive 33 deep mitochondrial lineages. Only seven of these corresponded to named species leaving 26 lineages as operational taxonomic units (OTUs). In times of DNA based taxonomy and species delimitation algorithms taxonomists face new challenges of how to describe a species and to avoid oversplitting. In this dissertation I evaluate, based on a taxonomic revision of Calumma nasutum group, the significance of mitochondrial lineages for species delimitation and promote micro-computed tomography (micro-CT) as an additional tool for integrative taxonomy. The second part deals with the discovery of the phenomenon of widespread fluorescence in chameleons. Biofluorescence is only rarely found in land vertebrates, so far, but has been reported for several marine organisms where it is used, inter alia, for intraspecific communication. As chameleons also communicate visually, the fluorescent pattern might work as an additional signal for species recognition. Methods and Procedure To describe (or redescribe) species of the Calumma nasutum group, I followed an integrative taxonomic approach incorporating five lines of evidence (for details, see below): External Morphology, micro-CT-scans of the skulls, dice-CT scans of the hemipenes, mitochondrial gene sequences (ND2), and nuclear gene sequences (CMOS). In the Calumma nasutum group a number of species have remained poorly characterized, because their original descriptions date to over a century ago and lack precise locality data, or because the holotype is a juvenile specimen. These species were redescribed here using a combination of micro-CT scans and detailed study of external morphology. With the help of diagnostic characters of the skull, the old type specimens were matched to recently collected and sequenced specimens. Additionally, micro-CT scanning was used for the first time to produce 3-dimensional models of chameleon hemipenes. In preparation for scanning, each hemipenis was removed from the specimen and immersed in iodine solution for several days to enhance the contrast when X-raying this soft tissue. This method is called dice-CT, and the resulting scans provide a more objective and detailed illustration of the hemipenes than the conventional 2-dimensional drawings. Further, sequences of mitochondrial (ND2) and nuclear (CMOS) genes were analysed for most of the new species described here. To provide comparability, access, and fast taxonomic progress, all new species were registered at ZooBank with an LSID number, their sequences were uploaded to GenBank, and all taxonomic acts were published in open access journals. To study fluorescence in chameleons, we used a fluorimeter to measure the excitation and emission spectra and to calculate the quantum yield for the intensity of fluorescence. The distribution of the fluorescent tubercles was recorded with photographs under UV light illumination, and the bony origins of fluorescence were studied using micro-CT and Transmission Electron microscopy of histological sections. Conclusions The taxonomic part or this dissertation resulted in the description of eight new chameleon species, "Calumma emelinae", C. gehringi, C. juliae, C. lefona, "C. ratnasariae", C. roaloko, "C. tjiasmantoi", and C. uetzi (see chapter 3.1.2, 3.1.3, 3.1.4, 3.1.5), contributing nearly 4% of all known species of the family Chamaeleonidae. Furthermore, one species, C. radamanus (Mertens, 1933) was revalidated, five species, C. boettgeri (Boulenger, 1888), C. fallax (Mocquard, 1900), C. guibei (Hillenius, 1959), C. linotum (Müller, 1924), and C. nasutum (Duméril & Bibron, 1836) were redescribed, and the females of C. vatosoa Andreone, Mattioli, Jesu & Randrianirina, 2001 were described for the first time. Using an integrative taxonomic approach, I showed that current species delimitation algorithms based on mitochondrial gene sequences alone greatly overestimate the actual number of species. Micro-CT proved essential for analysing skull morphology, which resulted more appropriate for species delimitation than some highly variable external characters. Further, this tool enabled to find frontoparietal fenestrae, which are cranial openings of potential adaptive importance found only in chameleon species living at high elevations. Using novel dice-CT imaging, I also analysed hemipenial morphology in minute detail and described a new ornament, the “cornucula gemina”. Finally, the phenomenon of fluorescence in chameleons was discovered in species belonging to eight of the twelve chameleon genera. The optimal excitation wavelength is in the UV-A spectrum at 353 nm, emitting light with wavelengths from 360 nm to 500 nm, with a maximum at 433 nm (blue spectrum). We showed that the fluorescent patterns result from bony tubercles on the skull are species specific, sexually dimorphic, and occur especially in forest living species. Based on these findings, and also because the colour blue is a conspicuous signal in forest habitats, I hypothesize that chameleons use fluorescence as a constant signal for intraspecific communication supplementing their vibrant body-colour language. Further, as-of-yet unpublished examples suggest that fluorescence is more common in squamates and might be an interesting field for further studies.Hintergrund Madagaskar ist bekannt für seine Biodiversität und seine außergewöhnlich hohe Endemismusrate. Dies gilt vor allem für Chamäleons, da auf der Insel beinahe die Hälfte aller Arten weltweit vorkommt. Die Erforschung dieser Artenvielfalt begann mit dem berühmten Georges Cuvier, der die ersten madagassischen Chamäleons im Jahre 1829 beschrieb. Seit damals haben sich die Qualität der Artbeschreibungen und folglich auch das Artkonzept substanziell verändert. Beruhten die ersten Beschreibungen noch auf wenigen Merkmalen der äußeren Morphologie, so werden heutzutage die gesammelten Exemplare gewöhnlich erst sequenziert und ihre Artzugehörigkeit anhand von genetischen Abständen bewertet. Auf diese Weise stellte eine vorangegangene Studie fest, dass die Calumma-nasutum-Gruppe, die aus kleinen Chamäleons mit gewöhnlich einem Nasenfortsatz auf der Schnauze besteht, insgesamt 33 tiefe mitochondriale Linien enthält. Nur sieben davon gehörten zu bereits beschriebenen Arten, wodurch noch 26 Linien als operational taxonomic units (OTUs) übrig blieben. In Zeiten der DNA-geleiteten Taxonomie und der Verwendung von Algorithmen zur Artabgrenzung stehen Taxonomen neuen Herausforderungen gegenüber, eine Art zu beschreiben und ein sogenanntes „oversplitting“ zu vermeiden. In dieser Dissertation bewerte ich am Beispiel der Revision der Calumma-nasutum-Gruppe die Bedeutung von mitochondrialen Linien zur Artabgrenzung und stelle Mikrocomputertomografie (Mikro-CT) als zusätzliches Werkzeug der integrativen Taxonomie vor. Der zweite Teil handelt von der Entdeckung des Phänomens der weitverbreiteten Fluoreszenz bei Chamäleons. Biofluoreszenz wurde bisher nur selten bei Landvertebraten nachgewiesen, ist jedoch bei einigen Meeresorganismen gut bekannt, welche die Fluoreszenz unter anderem zur intraspezifischen Kommunikation nutzen. Da Chamäleons ebenfalls optisch kommunizieren, könnten die Fluoreszenzmuster als zusätzliches Signal zur Arterkennung dienen. Methoden und Vorgehensweise Um Arten der Calumma-nasutum-Gruppe (wieder) zu beschreiben, nutzte ich einen integrativen taxonomischen Ansatz mit fünf Beweislinien (siehe unten im Detail): Äußere Morphologie, Mikro-CT-Scans der Schädel, dice-CT-Scans der Hemipenisse, Sequenzen der mitochondrialen Gene und Sequenzen der Kerngene. Einige Arten der Calumma-nasutum-Gruppe waren bisher wenig charakterisiert, da ihre Originalbeschreibung über einhundert Jahre zurücklag und genaue Typuslokalitäten fehlten oder weil der Holotypus ein Jungtier ist. Diese Arten wurden unter Verwendung von mikro-CT-Scans und genauer Untersuchung der äußeren Morphologie wiederbeschrieben. Mit Hilfe diagnostischer Merkmale des Schädels wurden die alten Holotypen kürzlich gesammelten und sequenzierten Individuen zugeordnet. Zusätzlich wurden zum ersten Mal Mikro-CT-Scans genutzt, um dreidimensionale Modelle von Chamäleonhemipenissen zu entwerfen. Zur Vorbereitung des Scans wurde jeder Hemipenis vom Tier abgetrennt und in Lugolscher Lösung für mehrere Tage eingelegt, um den Kontrast dieses weichen Gewebes beim Röntgen zu erhöhen. Diese Methode nennt sich dice-CT und die resultierenden Scans bieten eine objektivere und detailliertere Darstellung des Hemipenis als die herkömmlichen zweidimensionalen Zeichnungen. Weiterhin wurden Sequenzen von mitochondrialen Genen (ND2) und Kerngenen (CMOS) für die meisten der hier neu beschriebenen Arten analysiert. Um Vergleichbarkeit, weltweiten Zugang und schnellen taxonomischen Fortschritt zu garantieren, wurden alle neuen Arten bei ZooBank unter einer LSID-Nummer registriert, ihre Sequenzen bei GenBank hochgeladen und alle taxonomischen Arbeiten in open-access-Journalen veröffentlicht. Zur Untersuchung der Fluoreszenz bei Chamäleons benutzten wir ein Fluorimeter, um die Anregungs- und Emissionsspektren zu messen und die Quantenausbeute zu berechnen. Die Verteilung der fluoreszierenden Tuberkel wurde mit Fotos unter UV-Beleuchtung dokumentiert und ihr knöcherner Ursprung mit Mikro-CT und Transmissionselektronenmikroskopie der histologischen Schnitten untersucht. Schlussfolgerungen Im taxonomischen Teil dieser Dissertation wurden acht Chamäleonarten neu beschrieben, die beinahe 4% aller bisher bekannten Arten der Familie Chamaeleonidae ausmachen: "Calumma emelinae", C. gehringi, C. juliae, C. lefona, "C. ratnasariae", C. roaloko, "C. tjiasmantoi", und C. uetzi (siehe Kapitel 3.1.2, 3.1.3, 3.1.4, 3.1.5). Weiterhin wurde eine Art, C. radamanus (Mertens, 1933), revalidiert. Es wurden fünf Arten wiederbeschrieben: C. boettgeri (Boulenger, 1888), C. fallax (Mocquard, 1900), C. guibei (Hillenius, 1959), C. linotum (Müller, 1924), und C. nasutum (Duméril & Bibron, 1836), und die Weibchen von C. vatosoa Andreone, Mattioli, Jesu & Randrianirina, 2001 wurden erstmals beschrieben. Mit Verwendung eines integrativ taxonomischen Ansatzes konnte ich zeigen, dass Algorithmen zur Artabgrenzung, die allein auf mitochondrialen Gensequenzen basieren, die eigentliche Anzahl an Arten deutlich überschätzen. Mikro-CT bewährte sich um die Schädelmorphologie zu untersuchen, welche sich besser zur Artabgrenzung eignete als die sehr variablen äußeren Merkmale. Außerdem konnten mit Hilfe dieses Werkzeugs auch Frontoparietalfenster gefunden werden. Diese Schädelöffnungen wurden nur bei Chamäleons aus Montangebieten nachgewiesen und könnten eine besondere Anpassung an den Lebensraum darstellen. Das neuartige Dice-CT-Verfahren ermöglichte die Hemipenismorphologie im kleinsten Detail zu untersuchen und auch ein neues Ornament, die “cornucula gemina”, zu beschreiben. Schließlich wurde bei Arten aus acht der zwölf Chamäleongattungen das Phänomen der Fluoreszenz entdeckt. Die optimale Anregungswellenlänge liegt im UV-A-Spektrum bei 353 nm und Licht mit Wellenlängen von 360 bis 500 nm, mit einem Maximum bei 433 nm im blauen Spektrum, wird emittiert. Wir konnten zeigen, dass die Fluoreszenzmuster von knöchernen Tuberkeln des Schädels stammen, artspezifisch und sexualdimorph sind und vor allem bei waldbewohnenden Arten vorkommen. Darauf aufbauend und auch, da die Farbe Blau im Wald ein auffallendes Signal darstellt, formuliere ich die Hypothese, dass Chamäleons die Fluoreszenz als konstantes Signal zur intraspezifischen Kommunikation nutzen und damit ihre Farbensprache ergänzen. Nicht-publizierte Beispiele lassen außerdem vermuten, dass Fluoreszenz bei Squamaten weiter verbreitet ist und ein interessantes Gebiet für nachfolgende Studien darstellen könnte

Widespread bone-based fluorescence in chameleons

Fluorescence is widespread in marine organisms but uncommon in terrestrial tetrapods. We here show that many chameleon species have bony tubercles protruding from the skull that are visible through their scales, and fluoresce under UV light. Tubercles arising from bones of the skull displace all dermal layers other than a thin, transparent layer of epidermis, creating a 'window' onto the bone. In the genus Calumma, the number of these tubercles is sexually dimorphic in most species, suggesting a signalling role, and also strongly reflects species groups, indicating systematic value of these features. Co-option of the known fluorescent properties of bone has never before been shown, yet it is widespread in the chameleons of Madagascar and some African chameleon genera, particularly in those genera living in forested, humid habitats known to have a higher relative component of ambient UV light. The fluorescence emits with a maximum at around 430 nm in blue colour which contrasts well to the green and brown background reflectance of forest habitats. This discovery opens new avenues in the study of signalling among chameleons and sexual selection factors driving ornamentation

Splitting and lumping: An integrative taxonomic assessment of Malagasy chameleons in the Calumma guibei complex results in the new species C. gehringi sp. nov.

Calumma guibei (Hillenius, 1959) is a high-altitude chameleon species from the Tsaratanana massif in north Madagascar. Since its description was based on a juvenile holotype, its taxonomic identity is uncertain and little is known about its morphology. A recent molecular study discovered several deep mitochondrial clades in the Tsaratanana region assigned to C. guibei and C. linotum (Müller, 1924). In this paper we study the taxonomy of these clades and clarify the identity of C. guibei. Using an integrative taxonomic approach including pholidosis, morphological measurements, osteology, and molecular genetics we redescribe C. guibei and describe the new species C. gehringi sp. nov. which comprises two deep mitochondrial lineages. In terms of external morphology the new species differs from C. guibei by an elevated rostral crest, the shape of the notch between the occipital lobes (slightly connected vs. completely separated), presence of a dorsal and caudal crest in males (vs. absence), and a longer rostral appendage in the females. Additionally, we analysed skull and hemipenis morphology using micro-X-ray computed tomography (micro-CT) scans and discovered further differences in skull osteology, including a large frontoparietal fenestra, and separated prefrontal fontanelle and naris in C. guibei. Furthermore, we provide a comparison of micro-CT scans with traditional radiographs of the skull. The hemipenes have ornaments of two pairs of long pointed cornucula gemina (new term), two pairs of dentulous rotulae, and a pair of three-lobed rotulae, and are similar in both species, but significantly different from other species in the C. nasutum group. Geographically, C. guibei has been recorded reliably from the higher elevations of the Tsaratanana Massif above 1580 m a.s.l., whereas C. gehringi sp. nov. is found at mid-altitude (730–1540 m a.s.l.) in Tsaratanana and the surrounding area

Calumma boettgeri Boulenger 1888

Identity of <i>Calumma boettgeri</i> (Boulenger, 1888) <p> Morphological measurements and pholidosis of the holotype require the assignment of <i>Chamaeleo macrorhinus</i> to the Nosy Be form of <i>Calumma boettgeri.</i> The measurements are similar to the mean values of the females from Nosy Be (Tables 1–2): diameter of the broadest tubercle on the upper arm 0.34 mm (vs. mean of Nosy Be females of 0.32 mm, SD 0.048), number of enlarged tubercles on the upper arm 8 (vs. 10.6, SD 1.8); ratio of the upper arm diameter to the body size, 0.046 (vs. 0.041, SD 0.0038), total length, 93.1 mm (vs. 96.3 mm, SD 8.1 mm) and length of the rostral appendage, 2.8 mm (vs. 2.7 mm, SD 0.5 mm).</p> <p> <b>Diagnosis</b>. A small-sized chameleon (SVL 41.1–55.5 mm, TL 83.8–108.0 mm) that is characterised by a soft dermal, distally rounded, typically brown rostral appendage, slightly notched occipital lobes, the absence of a parietal crest, a low casque, the absence of axillary pits, small rounded tubercles not bordering each other on the extremities, presence or absence of a dorsal crest in males, low casque, and absence of gular and ventral crests. It differs from <i>C. guibei</i> by unnotched or only slightly (max. 0.7 mm) notched versus completely notched occipital lobes. For a distinction from <i>C. linotum</i>, see above.</p> <p> <b>Colouration in life.</b> The body and head colouration of males in relaxed state ranges from light brown to yellow without any obvious colour patterns. When stressed, dark colour patterns become prominent and the tail becomes annulated. A dark line runs from the snout tip across the eyes to the occipital lobes. The skin around the mouth and the throat can be white. Remarkable is the inconspicuous brown colour of the rostral appendage. The extremities appear brown also, except for a few green or blue coloured tubercle scales (Fig. 2 B).</p> <p>The colouration of the females can vary from beige to a reddish or greenish brown ground colouration in a relaxed state. The rostral appendage and the extremities show the same colour as the body, except for a few green tubercle scales on the legs. In a stressed state, three parallel bright blue spots appear on the upper half of the eyelids.</p> <p> <b>Distribution.</b> All confirmed distribution records of <i>C. boettgeri</i> are confined to the biogeographic Sambirano region in northwest Madagascar. It was found in both primary rainforest (Lokobe) and secondary forests (near Andoany) of Nosy Be (Andreone <i>et al.</i> 2003), in Manongarivo (Rakotomalala 2002; Gehring <i>et al.</i> 2012), and on Nosy Komba (Hyde Roberts & Daly 2014). Additionally Nagy <i>et al</i>. (2012, suppl. Fig. 2) identified a population with similar gene sequences to <i>C. boettgeri</i> from a forest fragment locally known as Maromiandra (13°99’65’’S, 48°21’77’’E, 283 m). According to molecular phylogenetic data, <i>C. boettgeri</i> from Nosy Be and one individual from Manongarivo (FGMV 2002-813, 13°58’62’’S, 48°25’32’’E, 751 m a.s.l.) form their own clade (Gehring <i>et al.</i> 2012). The elevations of all these localities range from 0 to 751 m a.s.l. (Fig. 6).</p> <p> We consider all additional records of <i>C. boettgeri</i>, all located in northeastern Madagascar, as in need of confirmation: Ambolokopatrika, 810–860 m a.s.l. (Andreone <i>et al.</i> 2000), Antalaha (Brygoo 1971), Andrakaraka forest station ca. 10 km from Antalaha (Ramanantsoa 1974), and Marojejy, 1100–1200 m a.s.l. (Raselimanana <i>et al.</i> 2000).</p>Published as part of <i>Prötzel, David, Ruthensteiner, Bernhard, Scherz, Mark D. & Glaw, Frank, 2015, Systematic revision of the Malagasy chameleons Calumma boettgeri and C. linotum (Squamata: Chamaeleonidae), pp. 211-231 in Zootaxa 4048 (2)</i> on pages 226-227, DOI: 10.11646/zootaxa.4048.2.4, <a href="http://zenodo.org/record/233651">http://zenodo.org/record/233651</a&gt

Systematic revision of the Malagasy chameleons Calumma boettgeri and C. linotum (Squamata: Chamaeleonidae)

Prötzel, David, Ruthensteiner, Bernhard, Scherz, Mark D., Glaw, Frank (2015): Systematic revision of the Malagasy chameleons Calumma boettgeri and C. linotum (Squamata: Chamaeleonidae). Zootaxa 4048 (2): 211-231, DOI: http://dx.doi.org/10.11646/zootaxa.4048.2.

Neon-green fluorescence in the desert gecko Pachydactylus rangei caused by iridophores

Abstract Biofluorescence is widespread in the natural world, but only recently discovered in terrestrial vertebrates. Here, we report on the discovery of iridophore-based, neon-green flourescence in the gecko Pachydactylus rangei, localised to the skin around the eyes and along the flanks. The maximum emission of the fluorescence is at a wavelength of 516 nm in the green spectrum (excitation maximum 465 nm, blue) with another, smaller peak at 430 nm. The fluorescent regions of the skin show large numbers of iridophores, which are lacking in the non-fluorescent parts. Two types of iridophores are recognized, fluorescent iridophores and basal, non-fluorescent iridophores, the latter of which might function as a mirror, amplifying the omnidirectional fluorescence. The strong intensity of the fluorescence (quantum yield of 12.5%) indicates this to be a highly effective mechanism, unique among tetrapods. Although the fluorescence is associated with iridophores, the spectra of emission and excitation as well as the small Stokes shifts argue against guanine crystals as its source, but rather a rigid pair of fluorophores. Further studies are necessary to identify their morphology and chemical structures. We hypothesise that this nocturnal gecko uses the neon-green fluorescence, excited by moonlight, for intraspecific signalling in its open desert habitat

Untangling the trees: Revision of the Calumma nasutum complex (Squamata: Chamaeleonidae)

Based on a large number of specimens and genetic samples, we revise the chameleons of the phenetic Calumma nasutum species group using an integrative taxonomic approach including external and hemipenial morphology, osteology, and sequences of a mitochondrial (ND2) and a nuclear gene (c-mos). After more than 180 years of taxonomic uncertainty, the eponymous species of the group, C. nasutum, is re-described, a lectotype is designated, and the species is assigned to a genetic clade that occurs in eastern (Anosibe An’Ala, Andasibe) and northern Madagascar (Sorata) based on morphology and osteology. The identity of C. fallax is also clarified and a lectotype is designated; it occurs at high elevation along the east coast from Andohahela (south) to Mandraka (central east). Calumma radamanus is resurrected from synonymy of C. nasutum ; it lives at low elevations in eastern Madagascar from Tampolo (south) to its type locality Ambatondradama (north). However, up to five deep mitochondrial lineages and high morphological variation are identified within C. radamanus, which we consider a species complex still in need of further taxonomic revision. Furthermore, three new species of the C. nasutum group are described: C. emelinae sp. nov. is distributed in eastern Madagascar (Anosibe An’Ala in the south to Makira in the north), C. tjiasmantoi sp. nov. in southeastern Madagascar (from Andohahela in the south to Ranomafana NP in the north), and C. ratnasariae sp. nov. is known from the Bealanana District in northern Madagascar. There is only little variation in hemipenial morphology in this group; the cornucula gemina are present in all taxa except the C. radamanus complex. Due to this taxonomic revision the protection status of the treated six chameleon species needs to be newly assessed; at least two of the species appear to warrant threatened statuses

Calumma boettgeri Prötzel, Ruthensteiner, Scherz & Glaw, 2015, sensu lato

Morphology of <i>Calumma boettgeri</i> sensu lato <p> <b>External morphology</b>. Measurements of important morphological parameters were taken from 23 specimens (11 males, 12 females) from Nosy Be, six specimens (five males, one female) from Montagne d’Ambre, and another nine specimens without exact locality data (Table 1). Because there was only one female from Montagne d’Ambre available, only males from both species were considered for comparison of body size, extremities and appendages. The specimens without localities were not included in mean value calculations but could be assigned according to their morphology to the Nosy Be morphotype (four specimens) and the Montagne d’Ambre morphotype (five specimens), respectively.</p> <p>, ratio of tail to snout-vent length; LRA, length of rostral appendage from snout tip; RRS, ratio of length of rostral appendage and snout-vent length; RAPSC, number of peripheral scales on rostral appendage; NPSCM, number of peripheral scales per mm on rostral appendage; RC, rostral crest present (+) or absent (-); NSL, number of supralabials; NIL, number of infralabials; OLD, lateral diameter of the occipital lobe; OLN, depth of the dorsal notch in occipital lobe; PC, parietal crest absent (-) or number of parietal cones; DC, dorsal crest absent (-) or number of dorsal cones; AP, axillary pits present (+) or absent (-); DSC, diameter of broadest scale on upper arm; RSB, ratio of broadest scale to snout-vent length; NSC, number of big scales on upper arm from lateral view; UAD, upper arm diameter; RAS, ratio of arm diameter to snout-vent length; all measurements in mm.</p> <p> <i>……continued on the next page …….continued on the next page</i> Continued.</p> <p> <b>collection no. species locality sex NIL OLD OLN PC DC AP DSC RSB NSC UAD RAS</b> <i>……continued on the next page</i> <b>collection no. species locality sex NIL OLD OLN PC DC AP DSC RSB NSC UAD RAS</b> SMF 16471 <i>C. boettgeri</i> Nosy Be f 10 4.2 0.0 - 0 - 0.4 0.007 9 2.2 0.044 SMF 16472 <i>C. boettgeri</i> Nosy Be f 13 3.5 0.1 - 11 - 0.4 0.009 10 1.8 0.039 ZSM 865/1920 <i>C. boettgeri</i> - f 13 2.8 0.1 - 0 - 0.3 0.006 9 1.9 0.038 ZFMK 51515 <i>C. boettgeri</i> - f 13 3.3 0.7 - 0 - 0.3 0.006 12 2.1 0.041 MCZ 5988 <i>Ch. macrorhinus</i> ‘Madagascar’ f 6.1 3.2 0.1 - 0 - 0.3 0.007 8 2.3 0.047 Individuals from Montagne d’Ambre show clear morphological differences from Nosy Be specimens (Fig. 7). The adult males examined from Montagne d’Ambre are larger than those from Nosy Be (mean values of TL 107.8 mm in Montagne d’Ambre vs. 99.3 mm in Nosy Be, Table 2), their rostral appendage is longer related to the snoutvent length (RRS 0.078 vs. 0.065) and the ratio of arm diameter to snout-vent length is larger (UAD/SVL 0.053 vs. 0.042).</p> <p> species <i>C. boettgeri C. boettgeri C. linotum</i></p> <p>locality Nosy Be Nosy Be M. d'Ambre</p> <p>sex m f m</p> <p>Both populations show heterogeneous scalation, especially at the extremities and the rostral appendage. The enlarged rounded tubercles on the limbs are distinctly larger in animals from Montagne d’Ambre; the mean diameter of the largest tubercle of the upper arm is 0.64 mm compared to 0.36 mm (Nosy Be). Additionally the number of the enlarged tubercles on the upper arm of males from Montagne d’Ambre is approximately twice that of males from Nosy Be, with a mean of 20.2 compared to 9.9, respectively, and the tubercles are not bordering each other on Nosy Be individuals. This character is also confirmed from a juvenile of SVL 26.9 mm (ZFMK 48227, Nosy Be) and a juvenile of SVL 36.2 mm (ZFMK 52308, Joffreville). Another morphological difference between both populations can be found in the pholidosis of the rostral appendage. Although the rostral appendage is significantly smaller in males from Nosy Be (see above), they have more peripheral scales on it, with a mean of 18.9 compared to 15.5 in Montagne d’Ambre males. In relation to the size of the appendage, this means 6.1 scales per mm compared with 4.1 scales per mm in Montagne d’Ambre. In summary, specimens from Montagne d’Ambre show a more heterogeneous scalation with broader tubercles on extremities and the rostral appendage. This is true of females as well (note that only one female with locality data was examined). Montagne d’Ambre individuals differ also in both sexes from Nosy Be specimens in the presence of a parietal crest which is best visible in the micro-CT scan (see below).</p> <p>The other morphological features either were highly variable or did not differ between the populations. For example, the number of dorsal cones was 0–28 in Nosy Be, and 9–13 in Montagne d’Ambre. Likewise the edges of the occipital lobes vary between the specimens. They range between un-notched and clearly notched (up to 0.7 mm) in Nosy Be, and are either not or only slightly notched (up to 0.3 mm) in Montagne d’Ambre. All specimens have a rostral crest and none have axillary pits.</p> <p> <b>Colouration.</b> Males also show great differences between the populations in colouration (see Fig. 2 A, B). Males from Montagne d’Ambre were more colourful, with a true blue rostral appendage and greenish turquoise extremities (Fig. 2 A). The colour of the legs is induced only by the coloured tubercle scales. The body is pale green or light brown with two dark brown spots and (occasionally) a beige lateral stripe on each side that stops at the base of the tail. The tail is the same colour as the body and (in stress colouration) possesses black annulations. The head is also greenish or brown with a dark stripe from the snout crossing the eyes to the occipital lobes. The skin around the mouth and the throat can be white. The colour description is based on a total of seven pictures of the Montagne d’Ambre form, referred to as <i>C. boettgeri</i> in Schmidt <i>et al.</i> (2010), <i>C. boettgeri</i> (picture 1c) in Glaw & Vences (2007) <i>Calumma</i> sp. in Nečas (2004) and <i>C. boettgeri</i> in Garbutt <i>et al.</i> (2001). The body and head of females is brown; the rostral appendage can be coloured bright blue, see picture of <i>C. boettgeri</i> in Schmidt <i>et al.</i> (2010).</p> <p> The body colouration of males from Nosy Be in contrast is yellowish or greenish brown with little dark brown rosettes, when stressed (Fig. 2 B). The legs are brown with little blue or green spots resulting from the tubercles. The colouration of the head is similar to the body colouration. The rostral appendage differs clearly from Montagne d’Ambre with the absence of any striking colour and is the same brown colour as the casque. Females are uniformly light or greenish brown coloured. Compare also a total of seven pictures of <i>Calumma boettgeri</i> in Hyde Roberts & Daly (2014), in Glaw & Vences (2007: 191) picture 1a and 1b, in Nečas (2004) and in Henkel & Schmidt (1995).</p> <p> <b>Osteology of the skull based on micro-CT scans.</b> Micro-CT scans of heads of two males and two females from Nosy Be and from Montagne d’Ambre exposed additional differences between the two forms. Specimens from Montagne d’Ambre (Fig. 4, D and E) bear tubercles on the parietal in both sexes. These form a little parietal crest in the middle with three to four tubercles, laterally followed by two tubercles on each side. The frontal is also irregularly spotted with tubercles. The parietal and frontal of animals from Nosy Be in contrast are smooth (Fig. 4, A and B).</p> <p> As in all species of the genus <i>Calumma</i>, the nasal bones are paired (Rieppel & Crumly 1997). These are broader in our specimens from Montagne d’Ambre (mean NW 0.35 mm vs. 0.24 mm in Nosy Be; mean NW/NL 0.18 vs. 0.14, Table 3, Fig. 4) and the anterior tip of the frontal bone does not exceed more than a half of the naris. In skulls from Nosy Be it does exceed this point, and the frontal meets the premaxilla, as described for <i>C. nasutum</i> (Rieppel & Crumly 1997). The parietal also varies between the two localities. In Nosy Be samples, the parietal tapers more tightly. Its diameter is at the tightest area on average 0.61 mm (vs. 1.06 mm) and 11% of the largest diameter of the parietal (vs. 22%, Table 3, Fig. 4). The parietal in Montagne d’Ambre samples appears wider and more compact. However, the form of the parietal is variable within localities and cannot be used as a diagnostic character. Although chameleons are sexually dimorphic animals, differences between sexes in skull structure were not proven (Table 3).</p> <p>Notes: m, male; f, female; NL, nasal length; NW, nasal width; RNWL, ratio of nasal width to length; PL, largest diameter; PS, parietal smallest diameter; RPSL, ratio of parietal smallest to largest diameter; PC, parietal crest absent (-) or number of tubercles.</p> <p> <b>Hemipenial morphology based on micro-CT scans.</b> The scans of hemipenes of specimens from each population enable a detailed view of their structure. The hemipenes are illustrated in sulcal and asulcal view with the apex on top (Fig. 5). Both populations show large and deep calyces with smooth ridges on the asulcal side of the truncus. The apex is ornamented with two pairs of long pointed papillae and two pairs of rotulae. The papillae rise from the sulcal side of the apex and are curved to the asulcal side. They can be completely everted (Fig. 5, C) or retracted in the apex (Fig. 5, D). One pair of rotulae is placed on the asulcal side (the smaller one) and one pair on the sulcal side. Here some differences between the populations are recognizable; in Nosy Be (n = 2) the rotulae are slightly more denticulated, with 6–11 tips on asulcal side and 14–16 tips on sulcal side, compared to Montagne d’Ambre (n = 3) with 6–8 tips on the rotulae of the asulcal side and 11–14 tips on both rotulae on the sulcal side.</p>Published as part of <i>Prötzel, David, Ruthensteiner, Bernhard, Scherz, Mark D. & Glaw, Frank, 2015, Systematic revision of the Malagasy chameleons Calumma boettgeri and C. linotum (Squamata: Chamaeleonidae), pp. 211-231 in Zootaxa 4048 (2)</i> on pages 214-221, DOI: 10.11646/zootaxa.4048.2.4, <a href="http://zenodo.org/record/233651">http://zenodo.org/record/233651</a&gt

Untangling the trees: Revision of the Calumma nasutum complex (Squamata: Chamaeleonidae)

Based on a large number of specimens and genetic samples, we revise the chameleons of the phenetic Calumma nasutum species group using an integrative taxonomic approach including external and hemipenial morphology, osteology, and sequences of a mitochondrial (ND2) and a nuclear gene (c-mos). After more than 180 years of taxonomic uncertainty, the eponymous species of the group, C. nasutum, is re-described, a lectotype is designated, and the species is assigned to a genetic clade that occurs in eastern (Anosibe An’Ala, Andasibe) and northern Madagascar (Sorata) based on morphology and osteology. The identity of C. fallax is also clarified and a lectotype is designated; it occurs at high elevation along the east coast from Andohahela (south) to Mandraka (central east). Calumma radamanus is resurrected from synonymy of C. nasutum ; it lives at low elevations in eastern Madagascar from Tampolo (south) to its type locality Ambatondradama (north). However, up to five deep mitochondrial lineages and high morphological variation are identified within C. radamanus, which we consider a species complex still in need of further taxonomic revision. Furthermore, three new species of the C. nasutum group are described: C. emelinae sp. nov. is distributed in eastern Madagascar (Anosibe An’Ala in the south to Makira in the north), C. tjiasmantoi sp. nov. in southeastern Madagascar (from Andohahela in the south to Ranomafana NP in the north), and C. ratnasariae sp. nov. is known from the Bealanana District in northern Madagascar. There is only little variation in hemipenial morphology in this group; the cornucula gemina are present in all taxa except the C. radamanus complex. Due to this taxonomic revision the protection status of the treated six chameleon species needs to be newly assessed; at least two of the species appear to warrant threatened statuses